The selective interaction between an aminoacyl-tRNA synthetase and its cognate tRNA is responsible for the fidelity of protein biosynthesis and represents a simple model system for examination of specific protein-nucleic acid interactions. A complete understanding of tRNA recognition at the molecular level requires a description of the three-dimensional crystal structure of tRNA synthetase-transfer RNA complexes. The goals of this project are: to prepare, characterize and crystallize nondissociable enzyme-tRNA complexes. The major experimental approach involves selective mercuration and "covalent" attachment of tRNAs to their cognate synthetases, which contain cysteinyl residues in their active site region. Secondly, structure and function studies of mammalian and bacterial enoyl-CoA hydratase, or crotonase, will be carried out in order to elucidate the mechanism of crotonase-catalyzed hydration, the effects of ligand binding on enzyme tertiary and quaternary structure and to identify active site amino acid residues. The results obtained and methods employed will serve as a model for further study of tRNA-recognition, and will increase our knowledge of the catalytic and regulatory mechanisms of protein biosynthesis and fatty acid beta-oxidation.